Stress-activated MAPK signaling pathways respond to a wide variety of stress conditions, including DNA damage, oxidative stress, heat shock, endoplasmic reticulum stress, hyper- and hypo-osmotic stress, shear stress, and a growing number of chemical toxins. Despite their organization with sensors at the cell surface connected to signaling components that typically culminate in the control of transcription factors and other proteins that impact cell physiology, there is growing evidence that many stressors activate these pathways through intracellular inputs rather than by signals that emanate from the cell surface. We have developed evidence in baker's yeast that a multitude of stress signals stimulate two stress-activated MAPK pathways, the Cell Wall Integrity (CWI) pathway and the High Osmolarity Glycerol (HOG) pathway, through intracellular inputs at various points along these pathways. This proposal is focused on identifying and characterizing the stress-specific components that feed into the MAPK cascades with a special focus on DNA damage-induced activation of the CWI pathway. Aim 1 extends our recent discovery that protein kinase C (Pkc1), which is the top protein kinase of the CWI MAPK cascade is a target of DNA damage checkpoint signaling. Pkc1 plays important roles in the response to and survival of DNA damage. Thus, we propose to take a combined phospho-proteomic, biochemical, and molecular genetic approach to understanding the impact of checkpoint signaling on the regulation of Pkc1 and the role of this pathway in the maintenance of genomic stability. Aim 2 seeks to identity stress-specific components involved in the intracellular activation of the CWI pathway MAPK cascade. These will focus on DNA damage, which activates the CWI MAPK (Mpk1) without the need for regulation of its upstream pathway components. This aim will also examine cell wall stress inputs, which surprisingly enter the pathway at a point within the MAPK cascade. We will use a combination of biochemical and mass spectrometric approaches, as well as genetic screens. Aim 3 is to identify and characterize intracellular stress inputs to the HOG MAPK cascade. The toxic metalloid arsenite and citric acid both activate the MAPK (Hog1) through an input that does not require regulation of upstream pathway components. Curcumin, a promising colon cancer and Alzheimer's therapeutic, signals to Hog1 through an input within the MAPK cascade. Completion of these aims will provide novel insights into the mechanisms by which stress signals activate MAPK pathways and will delineate a novel branch of the DNA damage response pathway.